Field of Invention
The invention generally relates to the improvement of the quality of analyte-staining on a biological tissue or cell sample that is affixed onto a solid substrate. More specifically, one or more embodiments of the invention relates to a method of biological sample treatment using a single multi-functional composition to perform multiple steps at an elevated temperature prior to staining of a target to remove paraffin, to rehydrate the deparaffinized sample, to retrieve targets on the sample, and the most importantly, to inhibit endogenous enzymes in the sample that may interfere with the staining. The staining method may be an analyte-staining method whereby an antibody is bound to its target antigen, or by an in situ hybridization method whereby a nucleic acid probe is bound to its target nucleic acid, such as DNA or RNA targets.
Background
Analyte-staining is a technique used to determine the presence of a target that is a protein or an antigen of interest, and to identify the pattern or the quantity of the protein expression in the context of tissue or cell morphology. When the sample source is a tissue sample, the analyte-staining technique is called immunohistochemistry (IHC); when the sample source is from cells, this staining technique is called immunocytochemistry (ICC).
In Situ Hybridization (ISH) is a method to determine the presence of a target that is DNA or RNA of interest, and to identify the pattern or the quantity of the DNA or RNA expression in the context of tissue.
This technology lays on the principle of the utilization of one or more probes that may be antibodies that specifically recognize and bind to the protein of interest, or the utilization of one or more probes that may be nucleotides that specifically recognize and bind to the DNA or RNA of interest. This is well-recognized as the step of “staining”. Being well-acknowledged and widely utilized in the field of art, the conventional or standard analyte-staining method is comprised of several preparation steps or pre-treatment steps that are sequentially performed prior to the staining steps.
For the analyte-assays that performed on tissue samples, the sample preparation steps include: 1) fixation of a biological sample, 2) embedding the sample into a matrix such as paraffin to solidify the sample, 3) cutting thin section of sample from the paraffin block and affixing the sections onto a microscope slide, 4) deparaffinization of the sample, 5) rehydration of the sample, 6) target retrieval, and 7) blocking endogenous enzymes present within the sample. Collectively these steps are preparation steps that are performed prior to the actual staining steps in order to prepare the samples for the optimal condition for the consequent staining step. Once the preparation steps have been completed the samples are ready for staining.
Under the situation that the tissue samples are freshly frozen, the above steps are modified accordingly to omit unnecessary steps including steps 2, 4 and 5.
When the sample source is a population of cells, the above steps is typically modified to omit unnecessary steps 2-5.
Formaldehyde-based fixatives are widely-used for fixation of the biological samples. Upon fixation, the aldehyde-based fixatives that have been widely used is generally believed in the field of art to cause cross-link reactions within and between the targets, which strengthen their structure; therefore preserving the morphology and the integrity of the sample. Solidification may further be achieved by freezing or paraffin embedding. The paraffin provides a firm matrix for tissue slicing. It is also considered the best option for long-term preservation of tissue samples.
The presence of paraffin or other equivalent matrix that solidify the sample, however, inhibits the subsequent staining process and results in a poor staining. Therefore, paraffin wax must be completely removed from the sample and the sample must be rehydrated. Deparaffinization is normally performed using organic solvent, such as xylene, toluene, d-limonene or other aliphatic hydrocarbon solvents. Standard histological methods for paraffin removal leave paraffin solvent in the biological sample. The paraffin solvent must be removed and replaced with water (or an aqueous solution) in a process called rehydration. Rehydration restores a sample to a more normal state conducive to molecular analysis.
Typically rehydration is a two-step process. The first step involves removing the paraffin solvent and replacing it with a water-miscible solvent. The ideal solvent for step one is any solvent that is both miscible with the paraffin solvent and with water. Such a solvent would be any alcohol, such as ethanol, methanol, or propanol. Other solvents could include acetone or methylene chloride. The most commonly used alcohol in histological laboratories is ethanol. The samples are incubated through a series of graded solutions starting with a mixture of the alcohol and paraffin solvent, gradually increasing the alcohol concentration until the samples are in pure alcohol. Following completion of this step the paraffin solvent is replaced by an alcohol, such as ethanol.
In the second step the alcohol is removed and replaced with water. The samples are incubated in a series of alcohol and water mixtures, gradually increasing the water concentration until the samples are in pure water. The water (or other aqueous solution) now permeates the sample, thus completing the rehydration process.
As an alternative, recent developments from various investigators have enabled that the deparaffinization be performed with an aqueous solution mixed with media that emulsifies. Upon heating, this aqueous solution would melt the paraffin while allowing the sample to retain in an aqueous environment at all times. Therefore, the next step of rehydration after deparaffinization using an organic solvent in the conventional analyte-staining method can be eliminated.
Paraffin inhibits the staining process, while the fixative solution halts all metabolic activity in the sample and immobilizes the molecular structure by altering their native structure or denaturing their original structure, likely upon creating chemical links within the molecular structure. Such links help maintain the molecular structure during staining and analysis. However such crosslinks may also denature the target by altering the molecular structure in such a way that the probes, antibodies or nucleic acids, can no longer bind to their targeted antigens, proteins, DNA or RNA. In this case the target is referred to as denatured. Denatured targets cannot be recognized by the probes, and the targets must first be retrieved and restored to their native configuration. The process of target retrieval is intended to retrieve targets such that they become recognizable by the probes. This process is alternatively well-recognized as unmasking or epitope unmasking.
There are generally two methods for target retrieval. The first method is termed enzyme antigen retrieval. In this method a proteolytic enzyme is applied to the biological sample and is allowed to moderately digest the protein structures such that the molecular crosslinks that cause target denaturing are broken and the targets are restored to a more natural configuration. The method of enzyme antigen retrieval is poorly understood and it is not clear why this method works for some kinds of targets but not for others. Some examples of proteolytic enzymes that have been shown to work for Enzyme Antigen Retrieval include pepsin, papain, trypsin, Proteinase K, Protease Type XXIV, and chymotrypsin. These enzymes in conventional use have an optimal temperature of 20° C.-37° C. Typically they are used at room temperature without heating. At temperature above 40° C., they lose their enzymatic activity. They are completely non-functional at temperatures of approximately 50° C. or higher. Therefore, when performed the samples are submerged in enzyme solution at a room temperature and allowed to incubate in this solution for about 5-10 minutes. The samples are then rinsed in buffer to complete the enzyme antigen retrieval step.
The second form of target retrieval is typically known as heat-induced antigen retrieval, often also referred to as heat-induced epitope retrieval (HIER). In this method the biological samples are heated to a temperature of about 90° C.-125° C., in an aqueous target retrieval solution. The heating of the sample results in the breaking of the methylene crosslinks caused by the fixation solution, thus restoring the denatured targets to a more natural configuration.
To date, enzyme target retrieval with proteolytic enzymes is rarely performed anymore because it has been shown that heat-induced target retrieval is generally superior. Occasionally the target retrieval is performed sequentially by using both enzyme target retrieval and heat target retrieval sequentially.
Previously it has not been possible to perform both methods of target retrieval simultaneously because the heating step inactivates the enzyme activity before it has had a chance to retrieve the targets.
The last key step prior to probe staining is endogenous enzyme blocking. Frequently the biological samples also contain the same enzymes used in the staining steps as naturally occurring products. Under these circumstances these naturally occurring enzymes are referred to as endogenous enzymes. When biological samples contain these endogenous enzymes, these enzymes must be inactivated prior to staining so that the endogenous enzymes do not interfere with the staining process. The endogenous enzymes that may be relied upon comprise peroxidase and alkaline phosphatase.
Up to date, for the target retrieval procedure, it has not been possible to perform both enzyme antigen retrieval method and heat-induced antigen retrieval method simultaneously because the heating step inactivates the enzyme activity before it has had a chance to retrieve the targets. Furthermore, for blocking, inhibition, or inactivation of an endogenous enzyme, up to date this step has been performed as a unique and individual step and has not been combined with other prior steps when preparing a sample for analyte-staining. Therefore, there is a need for improvement on the currently used analyte-staining methods with fewer steps. A single multi-functional aqueous composition that remains functional at an elevated temperature enables the combination of deparaffinization, rehydration, enzyme target retrieval, heat-induced target retrieval, and endogenous enzyme blocking. It simplifies the current staining assay method to combine multiple key procedures to one single step using one single solution prior to antibody staining, thus minimize possible operation errors either by hand or with automated settings.